Metallothioneins (MTs) are small proteins characterized by low molecular weight, high cysteinyl content; high capacity to bind Cd, Cu or Zn; highly conserved nucleic acid and amino acid sequences; and inducibility by diverse stimuli. Two isoforms exist in rodents. Repeated exposure to Cd aerosols causes a 400-fold augmentation in pulmonary MT. MT also accumulates in lung during exposure to 85 percent oxygen, albeit in smaller amounts. Animals adapt when repeatedly exposed to Cd. Cd-pretreated animals exhibit resistance to acute Cd challenge and are cross-tolerant to hyperoxia. The investigators hypothesize that MTs play a role in metal detoxification and free radical protection in the lung, and will evaluate the amplification of MT-mRNA during repeated Cd exposures by means of Northern analysis and dot blot hybridization techniques. MT isoforms will be separated and quantitated in lung tissue by HPLC in combination with a metal binding assay. The effect of MT transcriptional and translational status on the resistance of animals to acute Cd challenge will be assessed. Cd-induced cross tolerance to hyperoxia will be correlated to MT gene expression and alterations in lung cell populations, as determined by correlative light and electron microscopy and autoradiography. Differential pulmonary MT gene expression in Cd adapted and oxygen adapted rats will be compared. Type II epithelial cells, purified from rat lung by a cell """"""""panning"""""""" technique, and a pulmonary vascular endothelial cell line isolated from rat will be used to study mechanisms related to MT expression. MT induction will be assessed using various stimuli. MT gene products will be measured in lung cells using an ELISA procedure and by electrophoresis with Western blotting. MT isoform gene expression in type II cells, isolated from Cd and oxygen exposed animals will be determined. Coordinate induction of Md-SOD in these cells will be tested. The efficacy of MT as a protectant against free radicals will be assessed in vivo. The proposed studies hopefully will help to elucidate the function of MT in health and disease and contribute to our understanding of mechanisms which protect the lung against metal and free radical damage.
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